In order to sustain their prodigious anabolic needs tumors employ a specialized metabolism that differs from untransformed somatic cells. This metabolism leads to a tumor microenvironment (TME) that is acidic, hypoxic and depleted of critical nutrients required by immune cells. In this context tumor metabolism itself is a checkpoint that inhibits immune mediated tumor destruction. It stands to reason that targeting tumor metabolism might represent a potent means of ?Conditioning? tumors for killing by immunotherapy. Glutamine is essential for the aggressive growth characteristics of tumors. In collaboration with Dr. Barbara Slusher, head of the Johns Hopkins Drug Discovery Program, we have developed a novel agent to inhibit glutamine metabolism. The drug, JHU-083, is a pro-drug of the glutamine antagonist 6-diazo-5-oxo-l- norleucine (DON). DON blocks glutamine-utilizing reactions critical for the synthesis of nucleic acids, proteins and the generation of alpha-ketoglutarate. As a prodrug of DON, JHU-083 itself is inactive and is then converted in plasma by plasma esterases and intracellularly by cathepsin which are enriched in tumors compared to normal cells. While DON can cause gut and bone marrow toxicity, JHU-083 at similar doses avoids these side effects because its conversion to DON is enriched in the tumor. In this proposal, using JHU-083 as a tool to inhibit tumor metabolism, we will test the hypothesis that inhibiting tumor glutamine metabolism will not only inhibit tumor growth but will render tumors more susceptible to killing by immunotherapy. Our preliminary data demonstrate that treatment of tumors with JHU-083 changes the TME such that it is less acidic, less hypoxic and more replete with nutrients leading to enhanced endogenous anti-tumor responses and markedly enhancing the efficacy of immunotherapy in the form of checkpoint blockade, ACT and A2aR blockade. Furthermore, our preliminary studies demonstrate at the doses employed to inhibit tumor growth and change the TME, JHU-083 also acts on TILs to inhibit exhaustion and oxidative stress and enhance productive activation and memory generation. Based on these preliminary studies we will pursue the following specific Aims: 1. We will test the hypothesis that targeting tumor glutamine metabolism will change the tumor microenvironment (TME) such that the tumor will be more susceptible to immune mediated destruction. 2. We will test the hypothesize that treating mice with JHU-083 will enhance the efficacy of immunotherapy in the form of checkpoint blockade and ACT. 3. We will test the hypothesis that inhibition of tumor glutamine metabolism with JHU-083 will enhance the efficacy of immunotherapy in the form of A2Ar antagonism. At the completion of these studies we will have defined a novel approach to enhance anti-tumor immunotherapy. Furthermore, our studies will provide important insight into the role of tumor metabolism as a hurdle to the efficacy of immunotherapy. Finally, our findings provide the preclinical rationale for the development of DON prodrugs as a novel means of treating cancer.